This invention relates to enzymatic processes for converting glucose (dextrose) to fructose (levulose).
Most food grade glucose is provided as an enzymatic hydrolysate of corn starch, i.e., the corn syrup of commerce. Glucose is generally rated at being 60 to 80% as sweet as sucrose and therefore sells at a correspondingly lower price. It has long been known to isomerize glucose to fructose (which is even sweeter than sucrose) employing an enzyme having glucose isomerase activity, preferably one which has been immobilized upon an inert support such as diethylaminoethyl-cellulose, porous glass or chitin. Detailed descriptions of the enzymatic conversion of glucose to fructose employing glucose isomerase can be found in Hamilton, et al. "Glucose Isomerase a Case Study of Enzyme-Catalysed Process Technology", Immobilized Enzymes in Food and Microbial Processes, Olson et al., Plenum Press, New York, (1974), pp. 94-.+-.06, 112, 115-137; Antrim, et al., "Glucose Isomerase Production of High-Fructose Syrups", Applied Biochemistry and Bioengineering, Vol. 2, Academic Press (1979); Chen, et al., "Glucose Isomerase (a Review)", Process Biochem., (1980), pp. 30-35; Chen, et al. "Glucose Isomerase (a Review" , Process Biochem., (1980), pp. 36-41; Nordahl, et al., "Fructose Manufacture from Glucose by Immobiled Glucose Isomerase", Chem. Abstracts, Vol. 82, (1975), Abs. No. 110316h; and Takasaki, "Fructose Production Glucose Isomerase", Chem. Abstracts, Vol. 82, (1975), Abs. No. 110316h; and Takasaki, "Fructose Production by Glucose Isomerase", Chem. Abstracts, Vol. 81, (1974), Abs. No. 76474a. In addition, there are numerous patents relating to glucose isomerization of which U.S. Pat. Nos. 3,616,221; Reissue No. 28,885 (originally U.S. Pat. No. 3,623,953); 3,694,313; 3,708,397; 3,715,276; 3,788,945; 3,826,714; 3,843,442; 3,909,354, 3,960,663; 4,144,127; and, 4,308,349 are representative.
The levels of fructose achievable by the isomerization of glucose with glucose isomerase is limited by the equilibrium of the isomerase reaction. At 65.degree. C., the equilibrium of the reaction stands at approximately 51% fructose by weight from a starting substrate of pure dextrose. When refined glucose liquor is used as the substrate (containing up to about 6% nonmonosaccharides by weight) and allowing for a reasonable residence time in the enzyme reactor, an approximately 50% fructose syrup is the highest fructose content which can be obtained (on a dry basis) by the prior procedures referred to. To attain syrups of higher fructose content, fractionation systems must be employed which add greatly to the cost of the final product. At higher temperatures, however, the equilibrium becomes more favorable. For example, an enzymatic glucose isomerase process capable of being operated at temperatures of from about 90.degree.-130.degree. C. could be used to directly provide high fructose corn syrups (HFCS) containing 53-60 weight percent fructose on a dry basis thereby eliminating the need for fractionation and recycle. However, the tendency of known glucose isomerase systems to undergo thermal denaturation with an accompanying sharp reduction in activity has thus far frustrated attempts to utilize higher temperature regimes to force the equilibrium of the isomerization further in favor of fructose. Moreover, glucose and especially fructose are sensitive reducing sugars which have a marked tendency to form unwanted byproducts such as psicose, colored products, color precursors and acids when heated to the temperatures necessary to isomerize according to this invention. It has been speculated that the primary cause of thermal denaturation of proteins in general is the unfolding of the polypeptide chain and that such denaturation can be sharply retarded provided the protein globule is rigidified by attachment to a relatively rigid support in a multipoint fashion as described in Martinek, et al. Biochimica et Biophysica Acta, 485 (1977) 1-12, Elseview/North-Holland Biomedical Press. Torchilin, et al., Biochimica et Biophysica Acta, 522 (1978) 277-283, Elsevier/North-Holland Biomedical Press, reports a marked increase in the thermostability of alpha-chymotrypsin and succinylated alpha-chymotrypsin resulting from intramolecular crosslinkages achieved by activating the carboxylic acid groups of the enzyme with 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide followed by treatment with a diamine such as tetramethylenediamine. The authors do not state to what extent, if any, enzyme activity is affected by such crosslinking.
It is known from Japanese Patent NS No. 48-1181 to treat microbial cells containing glucose isomerase with the dialdehyde, glutaraldehyde, to bridge, or crosslink, the enzyme molecules. According to U.S. Pat. No. 3,796,634, extracellular glucose isomerase is first adsorbed on colloidal particles and thereafter treated with glutaraldehyde. U.S. Pat. No. 4,144,127 describes an immobilization procedure in which glucose isomerase is adsorbed upon colloidal silica followed by addition thereto of a bifunctional agent such as glutaraldehyde. U.S. Pat. No. 3,669,841 describes a general procedure for immobilizing enzymes, e.g., an isomerase, wherein the enzyme is linked to a siliceous carrier employing a crosslinking agent such as a dialdehyde of 2 to 8 carbon atoms of which glyoxal, glutaraldehyde, malonic aldehyde and succinaldehyde are representative.